Parasitology Research

, Volume 111, Issue 4, pp 1673–1682 | Cite as

Functional expression and characterization of an iron-containing superoxide dismutase of Acanthamoeba castellanii

  • Jung-Yeon Kim
  • Byoung-Kuk Na
  • Kyoung-Ju Song
  • Mi-Hyun Park
  • Yun-Kyu Park
  • Tong-Soo Kim
Original Paper


Acanthamoeba spp. are free-living amoebae, but opportunistic infections of some strains of the organisms cause severe diseases such as acanthamoebic keratitis, pneumonitis, and granulomatous amoebic encephalitis in human. In this study, we identified a gene encoding iron superoxide dismutase of Acanthamoeba castellanii (AcFe-SOD) and characterized biochemical and functional properties of the recombinant enzyme. Multiple sequence alignment of the deduced amino acid sequence of AcFe-SOD with those of previously reported iron-containing SODs (Fe-SODs) from other protozoan parasites showed that AcFe-SOD shared common metal-binding residues and motifs that are conserved in Fe-SODs. The genomic length of the AcFe-SOD gene was 926 bp consisting of five exons interrupted by four introns. The recombinant AcFe-SOD showed similar biochemical characteristics with its native enzyme and shared typical biochemical properties with other characterized Fe-SODs, including molecular structure, broad pH optimum, and sensitivity to hydrogen peroxide. Immunolocalization analysis revealed that the enzyme localized in the cytosol of the trophozoites. Activity and expression level of the enzyme were significantly increased under oxidative stressed conditions. These results collectively suggest that AcFe-SOD may play essential roles in the survival of the parasite not only by protecting itself from endogenous oxidative stress but also by detoxifying oxidative killing of the parasite by host immune effector cells.


Paraquat Oxidative Stressed Condition Amplify Polymerase Chain Reaction Product Acanthamoebic Keratitis Acanthamoeba Castellanii 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This study was supported by grants of the National Institute of Health, Korea Centers for Disease Control and Prevention (2005-N00180-00, 2006-N00183-00, and 2007-N00319-00).


  1. Alsam S, Jeong SR, Sissons J, Dudley R, Kim KS, Khan NA (2006) Escherichia coli interactions with Acanthamoeba: a symbiosis with environmental and clinical implications. J Med Microbiol 55:689–694PubMedCrossRefGoogle Scholar
  2. Barker J, Brown MR (1994) Trojan horses of the microbial world: protozoa and the survival of bacterial pathogens in the environment. Microbiology 140:1253–1259PubMedCrossRefGoogle Scholar
  3. Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44:276–287PubMedCrossRefGoogle Scholar
  4. Bécuwe P, Gratepanche S, Fourmaux MN, van Beeumen J, Samyn B, Mercereau-Puijalon O, Touzel JP, Slomianny C, Camus D, Dive D (1996) Characterization of iron-dependent endogenous superoxide dismutase of Plasmodium falciparum. Mol Biochem Parasitol 76:125–134PubMedCrossRefGoogle Scholar
  5. Berger P, Papazian L, Drancourt M, La Scola B, Auffray JP, Raoult D (2006) Ameba-associated microorganisms and diagnosis of nosocomial pneumonia. Emerg Infect Dis 12:248–255PubMedCrossRefGoogle Scholar
  6. Boucher IW, Brzozowski AM, Brannigan JA, Schnick C, Smith DJ, Kyes SA, Wilkinson AJ (2006) The crystal structure of superoxide dismutase from Plasmodium falciparum. BMC Struct Biol 6:20PubMedCrossRefGoogle Scholar
  7. Bozue JA, Johnson W (1996) Interaction of Legionella pneumophila with Acanthamoeba castellanii: uptake by coiling phagocytosis and inhibition of phagosome–lysosome fusion. Infect Immun 64:668–673PubMedGoogle Scholar
  8. Breathnach R, Chambon P (1981) Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem 50:349–383PubMedCrossRefGoogle Scholar
  9. Bruchhaus I, Brattig NW, Tannich E (1992) Recombinant expression, purification and biochemical characterization of a superoxide dismutase from Entamoeba histolytica. Arch Med Res 23:27–29PubMedGoogle Scholar
  10. Bus JS, Gibson JE (1984) Paraquat: model for oxidant-initiated toxicity. Environ Health Perspect 55:37–46PubMedCrossRefGoogle Scholar
  11. Carvalho FR, Foronda AS, Mannis MJ, Höfling-Lima AL, Belfort R Jr, de Freitas D (2009) Twenty years of Acanthamoeba keratitis. Cornea 28:516–519PubMedCrossRefGoogle Scholar
  12. Cho MH, Na BK, Song KJ, Cho JH, Kang SW, Lee KH, Song CY, Kim TS (2004) Cloning, expression, and characterization of iron-containing superoxide dismutase from Neospora caninum. J Parasitol 90:278–285PubMedCrossRefGoogle Scholar
  13. Choi DH, Na BK, Seo MS, Song HR, Song CY (2000) Purification and characterization of iron superoxide dismutase and copper–zinc superoxide dismutase from Acanthamoeba castellanii. J Parasitol 86:899–907PubMedGoogle Scholar
  14. Dufernez F, Yernaux C, Gerbod D, Noel C, Chauvenet M, Wintjens R, Edgcomb VP, Capron M, Opperdoes FR, Viscogliosi E (2006) The presence of four iron-containing superoxide dismutase isozymes in trypanosomatidae: characterization, subcellular localization, and phylogenetic origin in Trypanosoma brucei. Free Radic Biol Med 40:210–225PubMedCrossRefGoogle Scholar
  15. Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13PubMedCrossRefGoogle Scholar
  16. Fridovich I (1986) Superoxide dismutases. Adv Enzymol 58:62–97Google Scholar
  17. Fridovich I (1995) Superoxide radical and superoxide dismutase. Annu Rev Biochem 64:97–112PubMedCrossRefGoogle Scholar
  18. Fridovich I (1998) The trail to superoxide dismutase. Protein Sci 7:2688–2690PubMedCrossRefGoogle Scholar
  19. Fritsche TR, Sobek D, Gautom RK (1998) Enhancement of in vitro cytopathogenicity by Acanthamoeba spp. following acquisition of bacterial endosymbionts. FEMS Microbiol Lett 166:231–236PubMedCrossRefGoogle Scholar
  20. Gaze WH, Morgan G, Zhang L, Wellington EM (2011) Mimivirus-like particles in acanthamoebae from sewage sludge. Emerg Infect Dis 17:1127–1129PubMedCrossRefGoogle Scholar
  21. Gratepanche S, Menage S, Touati D, Wintjens R, Delplace P, Fontecave M, Masset A, Camus D, Dive D (2002) Biochemical and electron paramagnetic resonance study of the iron superoxide dismutase from Plasmodium falciparum. Mol Biochem Parasitol 120:237–246PubMedCrossRefGoogle Scholar
  22. Iovieno A, Ledee DR, Miller D, Alfonso EC (2010) Enhancement of in vitro cytopathogenicity by Acanthamoeba spp. following acquisition of bacterial endosymbionts. Ophthalmology 117:445–452PubMedCrossRefGoogle Scholar
  23. Ismail SO, Paramchuk W, Skeiky YA, Reed SG, Bhatia A, Gedamu L (1997) Molecular cloning and characterization of two iron superoxide dismutase cDNAs from Trypanosoma cruzi. Mol Biochem Parasitol 86:187–197PubMedCrossRefGoogle Scholar
  24. Jackson SMJ, Cooper JB (1998) An analysis of structural similarity in the iron and manganese superoxide dismutases based on known structure and sequences. Biometals 11:159–173PubMedCrossRefGoogle Scholar
  25. Kang JM, Cheun HI, Kim J, Moon SU, Park SJ, Kim TS, Sohn WM, Na BK (2008) Identification and characterization of a mitochondrial iron-superoxide dismutase of Cryptosporidium parvum. Parasitol Res 103:787–795PubMedCrossRefGoogle Scholar
  26. Marciano-Cabral F, Cabral G (2003) Acanthamoeba spp. as agents of disease in humans. Clin Microbiol Rev 16:273–307PubMedCrossRefGoogle Scholar
  27. Marciano-Cabral F, Toney DM (1998) The interaction of Acanthamoeba spp. with activated macrophages and with macrophage cell lines. J Eukaryot Microbiol 45:452–458PubMedCrossRefGoogle Scholar
  28. Meshnick SR, Eaton JW (1981) Leishmanial superoxide dismutase: a possible target for chemotherapy. Biochem Biophys Res Commun 102:970–976PubMedCrossRefGoogle Scholar
  29. Nogueira FB, Krieger MA, Nirde P, Goldenberg S, Romanha AJ, Murta SM (2006) Increased expression of iron-containing superoxide dismutase-A (TcFeSOD-A) enzyme in Trypanosoma cruzi population with in vitro-induced resistance to benznidazole. Acta Trop 100:119–132PubMedCrossRefGoogle Scholar
  30. Odberg-Ferragut C, Renault JP, Viscogliosi E, Toursel C, Briche I, Engels A, Lepage G, Morgenstern-Badarau I, Camus D, Tomavo S, Dive D (2000) Molecular cloning, expression analysis and iron metal cofactor characterisation of a superoxide dismutase from Toxoplasma gondii. Mol Biochem Parasitol 106:121–129PubMedCrossRefGoogle Scholar
  31. Ohno Y, Gallin JI (1985) Diffusion of extracellular hydrogen peroxide into intracellular compartments of human neutrophils. Studies utilizing the inactivation of myeloperoxidase by hydrogen peroxide and azide. J Biol Chem 260:8438–8446PubMedGoogle Scholar
  32. Paramchuk WJ, Ismail SO, Bhatia A, Gedamu L (1997) Cloning, characterization and overexpression of two iron superoxide dismutase cDNAs from Leishmania chagasi: role in pathogenesis. Mol Biochem Parasitol 90:203–221PubMedCrossRefGoogle Scholar
  33. Parker MW, Blake CC (1998) Iron- and manganese-containing superoxide dismutases can be distinguished by analysis of their primary structures. FEBS Lett 229:377–382CrossRefGoogle Scholar
  34. Saeki K, Hayakawa S, Isemura M, Miyase T (2000) Importance of a pyrogallol-type structure in catechin compounds for apoptosis-inducing activity. Phytochemistry 53:391–394PubMedCrossRefGoogle Scholar
  35. Scheid PL, Schwarzenberger R (2011) Free-living amoebae as vectors of cryptosporidia. Parasitol Res 109:499–504PubMedCrossRefGoogle Scholar
  36. Scheid P, Schwarzenberger R (2012) Acanthamoeba spp. as vehicle and reservoir of adenoviruses. Parasitol Res. doi: 10.1007/s00436-012-2828-7
  37. Scheid P, Zöller L, Pressmar S, Richard G, Michel R (2008) An extraordinary endocytobiont in Acanthamoeba sp. isolated from a patient with keratitis. Parasitol Res 102:945–950PubMedCrossRefGoogle Scholar
  38. Schuster FL (2002) Cultivation of pathogenic and opportunistic free-living amebas. Clin Microbiol Rev 15:342–354PubMedCrossRefGoogle Scholar
  39. Tannich E, Bruchhaus I, Walter RD, Horstmann RD (1991) Pathogenic and nonpathogenic Entamoeba histolytica: identification and molecular cloning of an iron-containing superoxide dismutase. Mol Biochem Parasitol 49:61–71PubMedCrossRefGoogle Scholar
  40. Temperton NJ, Wilkinson SR, Kelly JM (1996) Cloning of an Fe-superoxide dismutase gene homologue from Trypanosoma cruzi. Mol Biochem Parasitol 76:339–343PubMedCrossRefGoogle Scholar
  41. van Klink F, Taylor WM, Alizadeh H, Jager MJ, van Rooijen N, Niederkorn JY (1996) The role of macrophages in Acanthamoeba keratitis. Invest Ophthalmol Vis Sci 37:1271–1281PubMedGoogle Scholar
  42. Viscogliosi E, Delgado-Viscogliosi P, Gerbod D, Dauchez M, Gratepanche S, Alix AJ, Dive D (1998) Cloning and expression of an iron-containing superoxide dismutase in the parasitic protist, Trichomonas vaginalis. FEMS Microbiol Lett 161:115–123PubMedCrossRefGoogle Scholar
  43. Visvesvara GS, Moura H, Schuster FL (2007) Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea. FEMS Immunol Med Microbiol 50:1–26PubMedCrossRefGoogle Scholar
  44. Wassmann C, Hellberg A, Tannich E, Bruchhaus I (1999) Metronidazole resistance in the protozoan parasite Entamoeba histolytica is associated with increased expression of iron-containing superoxide dismutase and peroxiredoxin and decreased expression of ferredoxin 1 and flavin reductase. J Biol Chem 37:26051–26056CrossRefGoogle Scholar
  45. Wilkinson SR, Prathalingam SR, Taylor MC, Ahmed A, Horn D, Kelly JM (2006) Functional characterisation of the iron superoxide dismutase gene repertoire in Trypanosoma brucei. Free Radic Biol Med 40:198–209PubMedCrossRefGoogle Scholar
  46. Wilson CB, Tsai V, Remington JS (1980) Failure to trigger the oxidative metabolic burst by normal macrophages: possible mechanism for survival of intracellular pathogens. J Exp Med 151:328–346PubMedCrossRefGoogle Scholar
  47. Woyda-Ploszczyca A, Koziel A, Antos-Krzeminska N, Jarmuszkiewicz W (2011) Impact of oxidative stress on Acanthamoeba castellanii mitochondrial bioenergetics depends on cell growth stage. J Bioenerg Biomembr 43:217–225PubMedCrossRefGoogle Scholar
  48. Xuan YH, Yu HS, Jeong HJ, Seol SY, Chung DI, Kong HH (2007) Molecular characterization of bacterial endosymbionts of Acanthamoeba isolates from infected corneas of Korean patients. Korean J Parasitol 45:1–9PubMedCrossRefGoogle Scholar
  49. Yamada J, Yoshimura S, Yamakawa H, Sawada M, Nakagawa M, Hara S, Kaku Y, Iwama T, Naganawa T, Banno Y, Nakashima S, Sakai N (2003) Cell permeable ROS scavengers, Tiron and Tempol, rescue PC12 cell death caused by pyrogallol or hypoxia/reoxygenation. Neurosci Res 45:1–8PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Jung-Yeon Kim
    • 1
  • Byoung-Kuk Na
    • 2
  • Kyoung-Ju Song
    • 3
  • Mi-Hyun Park
    • 1
  • Yun-Kyu Park
    • 4
  • Tong-Soo Kim
    • 1
    • 4
  1. 1.Division of Malaria and Parasitic Diseases, National Institute of HealthKorea Centers for Disease Control and PreventionOsongSouth Korea
  2. 2.Department of Parasitology and Institute of Health SciencesGyeongsang National University School of MedicineJinjuSouth Korea
  3. 3.Department of Environmental Medical BiologyYonsei University College of MedicineSeoulSouth Korea
  4. 4.Department of Parasitology and Inha Research Institute for Medical SciencesInha University School of MedicineIncheonSouth Korea

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